Objective To construct the expression vector for ScFv against SOST and analyze antigen binding activity of ScFv. Methods Total RNA was extracted from hybridoma cell line 5H3D1 secreting mAbs against ScFv and CDNA was amplified by retropolymerase chain reaction ( RT-PCR). VL and VH were linked via a linker to construct ScFv ( VL-Linker-VH) gene by SOE-PCR. The ScFv gene was cloned into vector and expressed in HEK293 cells. The expressed proteins were detected by SDS-PAGE and antigen binding activity was analyzed by ELISA and Western blotting. The influences of ScFv on BMSCs differentiation were investigated by alizarin red S ( ARS) staining. Results The VL gene contained 339 base pairs and encoded 113 amine acid residues. The VH gene contained 330 base pairs and encoded 110 amine acid residues. There were four FRs, three CDRs and two characteristic cysteine residues in the VH gene and the VL gene, respectively. The ScFv gene contained 687 bases pairs and encode 229 amine acid residues with the structure of VL-linker- VH. ELISA analysis and Western blotting showed that the expression protein had specific antigen binding activity. The ScFv improve mineralized nodules formation. Conclusions The expression vector for ScFv against SOST has been successfully constructed to express protein having specific SOST binding activity. It has satisfactory effect on improving in vitro osteogenic potential of BMSc and establishes the basis for the treatment of osteoporosis with antibody.